We will continue studies of the initial steps of X inactivation, an essential developmental pathway for female sex defferenctiation. Inherent in our studies are insights into causes of the mental retardation and congenital malformations associated with the inability to inactivate specific genes. A region of the X chromosome, the X inactivation center(XIC), is required for its own inactivations (cis inactivation). There are good candidate genes (XIST and Xce) residing in the XIC, their roles have not been elucidated. We have uncovered X-chromosomal deletions (small ring chromosomes) which interfere with the chromosome's ability to inactivate, and result in severe congenital abnormalities. Now we want to identify sequences missing from these rings that are responsible for the failure to inactivate. These chromosomes with "defined function" deletions provide a unique analytical tool, (akin to adding or subtracting genes by molecular manipulation) for investigating cis-inactivation and the consequences of failure of dosage compensate. We will study the tiny ring chromosomes on hand and identify additional ones: rings (with breakpoints in the XIC region) which can or cannot inactivate. These chromosomes, isolated in hybrid cells or enriched by cell cloning, will be genotyped mainly using PCR analysis to identify breakpoint regions; interesting breakpoints will be refined using PFGE, FISH and Southern hybridization to narrow the XIC region to that essential for inactivation. We expect to identify sequences, in addition to XIST, that are required for cis inactivation. These could be analogous to Xce, enhancer of XIST function, or play a role independent of XIST expression, i.e., involved in X chromosome counting during embryogenesis. Identification of relevant sequences is facilitated by human YAC contigs and a library which includes cosmids from the region. Comparing genomic restriction maps with those of YAC's and cosmids should reveal regions invariably present on chromosomes that can inactivate, but missing from those that cannot. Genes in the pertinent cosmids will be identified by screening cDNA libraries using direct selection and exon trapping, and possibly homology with mouse cDNAs. Relevant regulatory sequences will be identified by looking for DNaseI hypersensitive and enhancer trapping. We will develop functional assays for XIST expression and X chromosome counting using YAC transfection into permissive cells. These studies will provide a better description of the XIC region, and the means to eliminate some hypotheses, thus limiting those that warrant further testing. Characterizing these ring X chromosomes provides a means not only to identify sequences and/or genes essential for cis inactivation, but also to determine genotype-phenotype correlations in preparation for mapping and positional cloning of genes who unregulated expression produces the severe phenotypes.